Department of Anesthesiology and Critical Care Medicine, The Johns Hopkins University School of Medicine, Baltimore, United States.
Department of Ophthalmology, The Johns Hopkins University School of Medicine, Baltimore, United States.
Elife. 2022 Oct 20;11:e76063. doi: 10.7554/eLife.76063.
Functionally distinct subtypes/clusters of dorsal root ganglion (DRG) neurons may play different roles in nerve regeneration and pain. However, details about their transcriptomic changes under neuropathic pain conditions remain unclear. Chronic constriction injury (CCI) of the sciatic nerve represents a well-established model of neuropathic pain, and we conducted single-cell RNA-sequencing (scRNA-seq) to characterize subtype-specific perturbations of transcriptomes in lumbar DRG neurons on day 7 post-CCI. By using mice that selectively express an enhanced in DRG neurons, we established a highly efficient purification process to enrich neurons for scRNA-seq. We observed the emergence of four prominent CCI-induced clusters and a loss of marker genes in injured neurons. Importantly, a portion of injured neurons from several clusters were spared from injury-induced identity loss, suggesting subtype-specific transcriptomic changes in injured neurons. Moreover, uninjured neurons, which are necessary for mediating the evoked pain, also demonstrated cell-type-specific transcriptomic perturbations in these clusters, but not in others. Notably, male and female mice showed differential transcriptomic changes in multiple neuronal clusters after CCI, suggesting transcriptomic sexual dimorphism in DRG neurons after nerve injury. Using as a proof-of-principle, RNAscope study provided further evidence of increased in injured neurons after CCI, supporting scRNA-seq analysis, and calcium imaging study unraveled a functional role of in neuronal excitability. These findings may contribute to the identification of new target genes and the development of DRG neuron cell-type-specific therapies for optimizing neuropathic pain treatment and nerve regeneration.
功能不同亚型/簇的背根神经节 (DRG) 神经元可能在神经再生和疼痛中发挥不同的作用。然而，关于它们在神经病理性疼痛条件下转录组变化的细节仍不清楚。坐骨神经慢性缩窄性损伤 (CCI) 代表一种成熟的神经病理性疼痛模型，我们进行了单细胞 RNA 测序 (scRNA-seq)，以表征 CCI 后第 7 天腰椎 DRG 神经元中亚型特异性转录组的扰动。通过使用选择性在 DRG 神经元中表达增强型 的 小鼠，我们建立了一种高效的纯化过程，以富集神经元进行 scRNA-seq。我们观察到四个突出的 CCI 诱导簇的出现和损伤神经元中标记基因的丢失。重要的是，来自几个簇的一部分损伤神经元免于损伤诱导的身份丧失，这表明损伤神经元中存在特定于亚型的转录组变化。此外，介导诱发疼痛所必需的未受伤神经元在这些簇中也表现出细胞类型特异性转录组扰动，但在其他簇中则没有。值得注意的是，CCI 后雄性和雌性小鼠在多个神经元簇中表现出不同的转录组变化，这表明神经损伤后 DRG 神经元中存在转录组性别二态性。使用 作为原理证明，RNAscope 研究进一步提供了 CCI 后 增加的证据，支持 scRNA-seq 分析，钙成像研究揭示了 在神经元兴奋性中的功能作用。这些发现可能有助于鉴定新的靶基因，并开发针对 DRG 神经元细胞类型的治疗方法，以优化神经病理性疼痛治疗和神经再生。
